Caenorhabditis elegans - PLOS

RESEARCH ARTICLE

Ilex paraguariensis modulates fat metabolism in Caenorhabditis elegans through purinergic system (ADOR-1) and nuclear hormone receptor (NHR-49) pathways Marina Lopes Machado1, Leticia Priscilla Arantes1, Priscila Gubert2, Daniele Coradini Zamberlan1, Thayanara Cruz da Silva1, Ta´ssia Limana da Silveira1, Aline Boligon3, Fe´lix Alexandre Antunes Soares ID1*

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1 Departamento de Bioquı´mica e Biologia Molecular, Programa de Po´s-graduação em Ciências Biolo´gicas: Bioquı´mica Toxicolo´gica, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil, 2 Centro de Ciências Biolo´gicas e da Sau´de, Campus Reitor Edgard Santos, Universidade Federal do Oeste da Bahia, Barreiras, Bahia, Brazil, 3 Departamento da Farmaćia Industrial, Laborato´rio de Pesquisa Fitoquı´mica, Universidade Federal de Santa Maria, Santa Maria, Rio Grande do Sul, Brazil * [email protected]

OPEN ACCESS Citation: Machado ML, Arantes LP, Gubert P, Zamberlan DC, da Silva TC, da Silveira TL, et al. (2018) Ilex paraguariensis modulates fat metabolism in Caenorhabditis elegans through purinergic system (ADOR-1) and nuclear hormone receptor (NHR-49) pathways. PLoS ONE 13(9): e0204023. https://doi.org/10.1371/journal. pone.0204023 Editor: Myon-Hee Lee, East Carolina University, UNITED STATES Received: February 6, 2018 Accepted: September 1, 2018 Published: September 25, 2018 Copyright: © 2018 Machado et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The financial support for this study was provided by “Programa de Apoio a Nućleos Emergentes” (PRONEM) [grant number 16/ 25510000248-7] to FAAS and Coordenação de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES-PROEX) [Process: 23038.005848/2018-

Abstract Ilex paraguariensis is a well-known plant that is widely consumed in South America, primarily as a drink called mate. Mate is described to have stimulant and medicinal properties. Considering the potential anti-lipid effects of I. paraguariensis infusion, we used an extract of this plant as a possible modulator of fat storage to control lipid metabolism in worms. Herein, the I. paraguariensis-dependent modulation of fat metabolism in Caenorhabditis elegans was investigated. C. elegans were treated with I. paraguariensis aqueous extract (1 mg/ml) from L1 larvae stage until adulthood, to simulate the primary form of consumption. Expression of adipocyte triglyceride lipase 1 (ATGL-1) and heat shock protein 16.2, lipid accumulation through C1-BODIPY-C12 (BODIPY) lipid staining, behavioral parameters, body length, total body energy expenditure and overall survival were analyzed. Total body energy expenditure was determined by the oxygen consumption rate in N2, nuclear hormone receptor knockout, nhr-49(nr2041), and adenosine receptor knockout, ador-1(ox489) strains. Ilex paraguariensis extract increased ATGL-1 expression 20.06% and decreased intestinal BODIPY fat staining 63.36%, compared with the respective control group, without affecting bacterial growth and energetic balance, while nhr-49 (nr2041) and ador-1(ox489) strains blocked the worm fat loss. In addition, I. paraguariensis increased the oxygen consumption in N2 worms, but not in mutant strains, increased N2 worm survival following juglone exposure, and did not alter hsp-16.2 expression. We demonstrate for the first time that I. paraguariensis can decrease fat storage and increase body energy expenditure in worms. These effects depend on the purinergic system (ADOR-1) and NHR-49 pathways. Ilex paraguariensis upregulated the expression of ATGL-1 to modulate fat metabolism. Furthermore, our data corroborates with other

PLOS ONE | https://doi.org/10.1371/journal.pone.0204023 September 25, 2018

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Ilex paraguariensis modulates fat metabolism in Caenorhabditis elegans

31, grant number 0737/2018]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

studies that demonstrate that C. elegans is a useful tool for studies of fat metabolism and energy consumption.

1. Introduction A number of plants are used as complementary or alternative medicines in regular diets worldwide [1]. Ilex paraguariensis St. Hil. Var. paraguariensis (Aquifoliaceae), the yerba mate, is widely used in southern Brazil, northern Argentina, Paraguay, and Uruguay [2] as a drink called chimarrão, tererê, or mate. Its consumption has been popular for centuries because/ of its stimulant and medicinal properties [3]. The effects of the consumption of I. paraguariensis include central nervous system stimulation [4], increased antioxidant defense [5], antioxidant properties in vitro [6], and thermogenic properties [7]. The prevalence of obesity is increasing worldwide, and has drawn the attention of public health institutions, as it is commonly associated with various metabolic disorders such as hypertension, dyslipidemia, type II diabetes, and insulin resistance [8]. In 2011–2012, 34.9% of adults aged 20 years and over were obese in the United States of America [9], indicating the urgent need for new treatments. Many methods are used to treat obesity, most of which are pharmaceuticals, which can cause collateral effects, like psychiatric disorders, heart attack, and stroke [10], and are often associated with rebound weight gain and potential drug abuse [11]. In this context, natural extracts are potential alternatives to pharmaceuticals and should be investigated for new anti-obesity treatments or for nutraceutical prevention for obesity, as they often cause less adverse effects and are easily added to the diet [12]. Previous studies have reported that the main compound found in aqueous extracts of I. paraguariensis are methylxanthines. The primary methylxanthine is caffeine [13, 14], which is a thermogenic agent that acts through the adenosine receptor [15] to increase metabolic rates [16–18], induce fat oxidation [19–21], stimulate respiratory centers [16], and increase resting energy expenditure [22]. Herein, we studied the modulation of fatty acid metabolism by I. paraguariensis extract in vivo using Caenorhabditis elegans as an animal model. This nematode has been described as a widely accepted and used model organism for studies of a variety of biological processes and diseases that can be defined on a molecular basis, e.g., obesity and fat metabolism, because many proteins involved in the synthesis, oxidation, and transport of lipids are highly conserved between C. elegans and mammals [23]. In C. elegans, adenosine receptor ortholog (ADOR-1), nuclear hormone receptor (NHR49), and adipose triglyceride lipase (ATGL-1) can be studied to evaluate the purinergic system [24], the regulation of β-oxidation [25] rate-limiting genes, and fat mobilization from stored triglycerides (TAGs) [26], respectively. This study aimed to investigate whether these pathways are involved in I. paraguariensis modulation of fat metabolism in C. elegans.

2. Materials and methods 2.1. Chemical, apparatus and general procedures of analytical grade Methanol, formic acid, gallic acid, chlorogenic acid and caffeic acid were purchased from Merck (Darmstadt, Germany). Quercetin, theobromine, caffeine, rutin, catechin, epigallocatechin and kaempferol were acquired from Sigma Chemical Co. (St. Louis, MO, USA). High performance liquid chromatography (HPLC-DAD) was performed with a Shimadzu Prominence Auto Sampler (SIL-20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with Shimadzu


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LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector and LC solution 1.22 SP1 software.

2.2. Plant material and aqueous extract preparation Minced leaves of Ilex paraguariensis from Ervateira Seiva-Pura1 used in this study were purchased from local market in Santa Maria, Rio Grande do Sul (Brazil). The extraction was carried out by pouring 100 mL of boiled distilled water on plant sample [27]. After extraction at room temperature (10 min), the aqueous extract was filtered using a sterilization filter with 0.22μm pore size.

2.3. Quantification of compounds by HPLC-DAD Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm x 250 mm) packed with 5μm diameter particles. The mobile phase was water containing 1% formic acid (A) and methanol (B), and the composition gradient was: 15% of B until 10 min and changed to obtain 20%, 30%, 50%, 60%, 70%, 20% and 10% B at 20, 30, 40, 50, 60, 70 and 80 min, respectively, following the method described by Abbas et al. (2014) with slight modifications [28]. Ilex paraguariensis aqueous extract was analyzed at a concentration of 20 mg/Mr. The presence of ten compounds was investigated: Gallic acid, chlorogenic acid, caffeic acid, catechin, epigallocatechin, quercetin, rutin, kaempferol, caffeine and theobromine. Identification of these compounds was performed by comparing their retention time and UV absorption spectrum with those of the commercial standards. The flow rate was 0.7 ml/min, injection volume 40 μL and the wavelength were 257 nm for gallic acid, 270 nm for theobromine, 280 nm for catechin, epigallocatechin and caffeine, 327 nm for caffeic and chlorogenic acids, and 366 nm for quercetin, rutin and kaempferol (Table 1). All the samples and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath prior to use. Stock solutions of standards references were prepared in the HPLC mobile phase at a concentration range of 0.030–0.250 mg/ml for kaempferol, quercetin, catechin, epigallocatechin, rutin, caffeine and theobromine; and 0.045–0.300 mg/ml for gallic, caffeic and chlorogenic acids. The chromatography peaks were confirmed by comparing its retention time with those of reference standards and by DAD spectra (200 to 600 nm). Table 1. Composition of Ilex paraguariensis aqueous extracts. Compounds

Ilex paraguariensis mg/g

LOD

LOQ

μg/mL

%

μg/mL

1.27 ± 0.01a

0.12

0.015

0.049

Catechin

b

2.98 ± 0.03

0.29

0.032

0.105

Chlorogenic acid

3.71 ± 0.01b

0.37

0.008

0.027

Caffeic acid

9.15 ± 0.02c

0.91

0.021

0.070

Caffeine

8.86 ± 0.01d

0.88

0.029

0.095

Theobromine

3.65 ± 0.01b

0.36

0.007

0.023

Epigallocatechin

6.01 ± 0.03e

0.60

0.016

0.052

Rutin

7.43 ± 0.02f

0.74

0.026

0.086

Quercetin

3.12 ± 0.01b

0.31

0.035

0.115

e

0.59

0.019

0.063

Gallic acid

Kaempferol

5.95 ± 0.03

Results are expressed as the mean ± standard deviation (SD) of three determinations. Averages followed by different letters differ by Tukey test at p < 0.05. LOD, limit of detection; LOQ, limit of quantification. https://doi.org/10.1371/journal.pone.0204023.t001


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Calibration curve for gallic acid: Y = 13057x + 1285.4 (r = 0.9998); catechin: Y = 12728x + 1197.5 (r = 0.9995); epigallocatechin: Y = 11893 + 1357.2 (r = 0.9995); chlorogenic acid: Y = 12659x + 1287.8 (r = 0.9993); caffeic acid: Y = 11962x + 1326.2 (r = 0.9997); caffeine: Y = 13276x + 1297.6 (r = 0.9999); theobromine: Y = 12473x + 1175.8 (r = 0.9996); rutin: Y = 13805 + 1195.7 (r = 0.9999); quercetin: Y = 13627x + 1362.1 (r = 0.9999) and kaempferol: Y = 12583x + 1274.8 (r = 0.9997). The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves. LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the standard deviation of the response and S is the slope of the calibration curve [29].

2.4. C. elegans strains Wild-type C. elegans strain N2 wild-type (var. Bristol), STE68 nhr-49(nr2041), VS20 (hjIs67 [atgl-1p::atgl-1::gfp + mec-7::RFP]) and CL2070 dvIs70 Is[hsp-16.2::gfp; rol-6(su1006)] were provided by the Caenorhabditis Genetics Center (University of Minnesota, USA). EG6870 strain, ador-1(ox489), was kindly supplied from Dr. Erik Jorgensen laboratory (University of Utah, USA). This strain has a deletion from 1kb upstream and the first three exons of the ador-1 gene, and was outcrossed six times. All strains were maintained at 20˚C.

2.5. Growth conditions and Ilex paraguariensis treatment Treatment plates were prepared diluting Ilex paraguariensis aqueous extract in distilled autoclaved water and spreading it with Escherichia coli OP50 as food source to the surface of dry nematode grow media (NGM) agar plates [30] to final concentrations of 0, 0.25, 0.5 and 1 mg/ mL. Control plates were prepared with water and bacteria at the same proportions. Plates were incubated overnight at 37˚C to allow bacteria growth. Synchronized L1 worms were cultured onto treatment plates in the presence or absence of aqueous extract and allowed to develop until the young adult stage at 20˚C.

2.6. Bacterial growth curve E. coli OP50 growth was evaluated over 4 h in the presence or absence of Ilex paraguariensis at 0.25, 0.5 or 1 mg/mL. The optical density was measured with a spectrophotometer at 600 nm. Growth curves were normalized with the control group at time zero [31].

2.7. ATGL-1::GFP expression To determine ATGL-1::GFP expression, young-adults worms from VS20 strain were immobilized with 10 mM sodium azide for image acquisition (Fig 1), and photographed under 60× objective on a confocal microscope (Fluoview FV101, Olympus, Tokyo, Japan). Images were processed with the Olympus Image Browser. GFP fluorescence quantification and analyses of images were conducted with ImageJ software by determining average pixel intensity per animal.

2.8. C1-BODIPY-C12 staining C1-BODIPY-C12 conjugated fatty acids (BODIPY) lipid staining was carried out as previously described [32]. C1-BODIPY-C12 was applied to the surface of NGM plates (10 mL agar) seeded with E. coli OP50 and 0 or 1 mg/mL of Ilex paraguariensis to a 50 nM final concentration (Fig 2 or S1 Fig). Synchronized wild-type (N2), nhr-49(nr2041), and ador-1(ox489) L1-stage worms were transferred to these plates and allowed to develop until adulthood.


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Fig 1. Ilex paraguariensis effects on ATGL-1::GFP expression in Caenorhabditis elegans VS20 strain. (A) Visualization of ATGL-1::GFP expression and (B) measurement of ATGL-1::GFP expression on young-adult Caenorhabditis elegans. p